A method is described for the routine preparation of sputum samples for detection of Mycobacterium tuberculosis by using polymerase chain reaction amplification. Liquefaction of sputum samples with NaOH and subsequent removal of inhibitors of the polymerase reaction with a 50% sucrose centrifugation step (5 min) in a desktop centrifuge allow direct amplification of a 123-bp repetitive region of the M. tuberculosis genome. We have evaluated the sensitivity and specificity of the sucrose method with 155 sputum specimens from patients suspected of having tuberculosis and from normal healthy volunteers. This method, in our opinion, is reproducible, sensitive, and reliable. Multiple samples can be handled simultaneously, and results can be obtained in one working day.